Brain functioning under acute hypothermic stress supported by dynamic monocarboxylate utilization and transport in ectothermic fish
© Tseng et al. 2014
Received: 18 February 2014
Accepted: 11 July 2014
Published: 8 August 2014
The vertebrate brain is a highly energy consuming organ that requires continuous energy provision. Energy metabolism of ectothermic organisms is directly affected by environmental temperature changes and has been demonstrated to affect brain energy balance in fish. Fish were hypothesized to metabolize lactate as an additional energy substrate during acute exposure to energy demanding environmental abiotic fluctuations to support brain functionality. However, to date the pathways of lactate mobilization and transport in the fish brain are not well understood, and may represent a critical physiological feature in ectotherms during acclimation to low temperature.
We found depressed routine metabolic rates in zebrafish during acute exposure to hypothermic (18°C) conditions accompanied by decreased lactate concentrations in brain tissues. No changes in brain glucose content were observed. Acute cold stress increased protein concentrations of lactate dehydrogenase 1 (LDH1) and citrate synthase (CS) in brain by 1.8- and- 2.5-fold, paralleled by an increased pyruvate to acetyl-CoA transformation. To test the involvement of monocarboxylate transporters (MCTs) under acute cold stress in zebrafish, we cloned and sequenced seven MCT1-4 homologues in zebrafish. All drMCT1-4 are expressed in brain tissues and in response to cold stress drmct2a and drmct4a transcripts were up-regulated 5- and 3-fold, respectively. On the contrary, mRNA levels of drmct1a, -1b and -4b in zebrafish brain responded with a down regulation in response to cold stress. By expressing drMCTs in Xenopus oocytes we could provide functional evidence that hypothermic stress leads to a 2-fold increase in lactate transport in drMCT4b expressing oocytes. Lactate transport of other paralogues expressed in oocytes was unaffected, or even decreased during cold stress.
The present work provides evidence that lactate utilization and transport pathways represent an important energy homeostatic feature to maintain vital functions of brain cells during acute cold stress in ectotherms.
Fluctuating environmental temperatures are a major stressor for ectothermic animals which can severely affect vital biochemical and physiological processes ,. Ectothermic organisms have evolved a range of mechanisms to maintain physiological functions during environmental temperature fluctuations ,,. In the context of hypothermic tolerance special attention has been dedicated to energy metabolism related processes. For example, in blood and liver of common eelpout (Zoarces viviparus), the levels of adenosine were elevated under cold stress via adenosine-triphosphate (ATP) hydrolysis indicating that immediate energy supply and the activation of associated enzymes are essential for ectothermic animals undergoing hypothermic acclimation . Furthermore the utilization of lactate to generate energy equivalents has been hypothesized to represent an essential adaptive mechanism to support cellular and organismic functionality during hypothermic stress. For example, Antarctic fish brains were demonstrated to have higher activities of lactate dehydrogenases (LDH) and citrate synthases (CS) when compared to tropical and subtropical species ,. Neuronal functions may thus remain regular and well-coordinated by maintaining metabolic rates in brain tissues during cold acclimation. For example, in isolated brain slices of teleost fishes, high oxygen consumption rates during cold acclimation have been reported ,. These results suggest that at least a partial compensation to cold stress may exist, which is fueled by aerobic ATP producing pathways in fish brain. Moreover, earlier studies using zebrafish could demonstrate a mild mitochondrial uncoupling resulting in enhanced heat production in brain during acute cold stress . Additionally increased activation of glucose transporter (GLUT) and ATP production via glycolysis has been suggested to allow for a metabolic shift to maintain physiological energy balance in brains of zebrafish under acute cold stress . Therefore it was concluded that despite reductions in metabolic rate on the whole animal level, cold acclimated/adapted fish need to maintain brain energy homeostasis by temperature-compensatory mechanism to support proper functionality. This suggests that brain tissues have evolved compensatory metabolic features that allow fish to maintain brain functionality in a hypothermic environment –.
The brain is a highly energy consuming organ and very sensitive to fluctuations in energy supply ,. Most energy consumed by brain cells is devoted to message signal transduction, systemic endocrine regulation, ionic gradient maintenance and restoration after depolarization –. The brain belongs to the central nervous system and is encased in fluid-filled meninges which consist of neurons, glial cells and capillaries separated from other tissues by a blood-brain barrier (BBB) ,. High concentrations of GLUTs, monocarboxylate transporters (MCTs) and excitatory amino acid transporters (EAATs), facilitate the transport of energy equivalents across membranes of diverse cell types that build up the central nervous system (CNS) ,,. Although glucose represents the most important energy substrate ,– a growing number of studies suggested that besides glucose oxidation lactate may also represent an important substrate to provide energy to brain cells ,,. The transport and utilization of lactate is based on the “astrocyte-neuron lactate shuttle hypothesis” and has already been proposed as a central energy providing pathway in the mammalian CNS ,. Lactate is transported from astrocytes to neurons through MCT1 and 4  and is converted to pyruvate by LDH1. Subsequently pyruvate dehydrogenase (E1) catalyzes the transformation of pyruvate to acetyl-CoA and enters the Krebs cycle to produce ATPs to fuel the energetic demands of neuron cells –. In this context special attention has been dedicated to monocarboxylate transporters (MCTs) that enable the transport of lactate in the mammalian brain ,,,. MCTs belong to the solute carrier family (SLC) 16, which consists of 14 members in mammals –. Only MCT1, MCT2, MCT3 and MCT4 have been demonstrated to be responsible for the trans-membrane transport of relevant monocarboxylic acids, such as lactate, pyruvate and ketone bodies ,,. In mammals, MCT1 can be found in membranes of neurons and astrocytes of the CNS . MCT1 was also detected in the sarcolemma of oxidative skeletal muscle fibers and cardiomyocytes, but never in fast-twitch glycolytic skeletal muscle fibers –. The lactate transport mechanism of MCT1 has been thoroughly studied ,. MCT1 binds one proton and undergoes a conformational change, which transports the lactate molecule across the membrane –. Another paralogue, MCT2 has been demonstrated to be the major player for monocarboxylate transport in rodent brain neurons ,–. Over-expression of MCT2 in Xenopus oocytes revealed that this protein is also driven by proton fluxes and has a higher affinity for monocarboxylates than MCT1. The high affinity of MCT2 for monocarboxylates makes it especially suitable for transporting lactate into neurons for oxidation as an important energy source . Moreover, MCT3 has a unique distribution in the basal membrane of retinal pigment epithelium and choroid plexus epithelia of mammals, but the detailed characterization of the transport kinetics have not been studied so far ,. MCT4 has been reported to be localized in astrocytes, fast-twitch oxidative glycolytic skeletal muscle fibers and cardiomyocytes ,. The lactate affinity of MCT4 is lower than that of MCT1 and MCT2, but MCT4 has a high capacity for lactate transport ,,. In summary, MCTs play an important role in the trafficking of lactate into energy-consuming cells to meet their energetic demands. Regarding lactate utilization in brains of teleosts, Polakof and colleagues , have demonstrated that lactate can act as an alternative energy fuel in glucose-sensing brain regions such as hypothalamus and hindbrain. Thus, in ectothermic fish brain, lactate may have a similar metabolic role in maintaining energy homeostasis as observed in mammals ,. However, the cellular mechanistic basis for lactate utilization and transport in brain of ectothermic animals are largely unexplored and may significantly differ from those described for mammalian systems. Zebrafish (Danio rerio) has been recently explored as a suitable model organism to study the effects of environmental stressors including hypothermic stress on various physiological processes ,–. Therefore, we use this ectothermic model species to study lactate utilization strategies and further demonstrate the role of MCTs in energy metabolism of brain under acute cold stress. First, we investigated the effect of cold acclimation on several metabolic indexes (O2 consumption and NH4+ excretion), carbohydrate contents and relevant metabolites enzymes to clarify if cold stress induced changes in metabolism are associated to enhanced lactate utilization. Based on our previous study that identified drMCT2 as an indispensable monocarboxylate-transporting route for brain development and function in zebrafish . In order to extend our knowledge regarding differential functions of MCT isoforms in fish during cold-acclimation we identified and characterized additional teleost-specific isoforms. We hypothesize an evolutionary trend for diversified functions and dynamic expression patterns of MCT isoforms in the brain of ectothermic fish (Tseng et al. ). We further asked if drMCTs are involved in lactate utilization during cold acclimation and if drMCT isoforms play differential roles in this process. Accordingly, temperature-dependent lactate transport of MCT homologues’ was characterized via expression and over-expression in Xenopus oocytes. These findings contribute to a better understanding for energy provision by monocarboxylate transport pathways during cold acclimation in brains of ectothermic vertebrates.
Routine metabolic rates under acute cold exposure
Glucose and lactate concentrations in zebrafish brain under acute hypothermic stress
Lactate levels in brain of zebrafish kept at 28°C was 48.33 ± 7.48 nmole/mg. In comparison to control (28°C) animals, lactate contents in zebrafish brains which were exposed to 18°C for 1 and 24 h were decreased to 26.66 ± 2.48 and 25.36 ± 1.95 nmole/mg, respectively (Figure 2B). This indicates a 45% to 49% decrease in brain lactate contents during 1 and 24 h 18°C exposure compared to control animals.
Protein concentration and transcript abundance of metabolic genes during hypothermic stress
Identification of drMCTs in zebrafish
Expression of drMCTs in brains of zebrafish
35 cycles amplification of RT-PCR analysis for various MCTs was conducted in different tissues including brain, gill, eye, spleen and liver from adult zebrafish. Except from drmct1b and -4b which were not detected in liver, drmct1a, -2a, -2b, -3 and -4a were ubiquitously expressed in various tissues of zebrafish. All the transcripts of drmct1-4 paralogues were detected in brain tissues (Figure 7A).
Based on our previous studies, spatial expressions of drmct1a, -2a, -3 and -4a mRNAs in brains of adult zebrafish were already proved to be expressed in both neurons and astrocytes . To further identify cell types that express three novel drMCT1b, -2b and -4b, in vitro synthesized RNA probes were used to detect mRNA of these paralogues in transverse sections of zebrafish brains hypothalamus. Subsequent immunocytochemical labeling with ZN12 and glial fibrillary acidic protein (GFAP) antibodies was carried out to specifically identify neurons and astrocytes, respectively ,. In our preliminary test, ZN12 and GFAP were shown to specifically characterize neurons and astrocytes, respectively, in the midbrain of zebrafish . On one hand as shown in Figure 7B and C, drmct1b and -2b (fluorescence in situ hybridization) were both detected in specified groups of brain cells, which were recognized as neuron cells with the ZN12 antibody but never co-localized with GFAP signals. On the other hand, drmct4b was detected in both ZN12 and GFAP recognized brain cells (Figure 7D). Therefore, these images indicated that drmct4b was expressed in both neurons and astrocytes.
In vitro functional analysis of drMCTs in Xenopus laevis oocytes
The present work demonstrated that lactate utilization in zebrafish brain represents an advantageous feature to adequately and timely provide energy in brain tissues during acute (1- and 24 h) cold exposure. Furthermore, drMCT isoforms expressed in oocytes reveal differential characteristics in terms of temperature-dependent lactate transport. The lactate uptake of drMCT2a expressing oocytes was decreased while those expressing drMCT4b were stimulated by low temperature (18°C). These findings, relevant for brain functionality during cold stress are discussed in the following and embedded in the existing body of knowledge.
In the present study, oxygen consumption rates of adult zebrafish transferred from 28°C to 18°C for 1 and 24 h were 2- to 6-fold decreased, indicating that acute hypothermic stress directly translates into decreased metabolic rates on the whole animal level. On one hand, hypothermic challenges may directly affect metabolic enzyme activities and muscle contraction; on the other hand, cold-induced activation of the neuroendocrine system (e.g. TRH release) may be beneficial to counteract physiological compromises during hypothermic stress . Similar observations were made for a range of other fish species with Q10 values between 2 and 3 although Q10 may show strong differences between species and ontogenetic stages –. The fact that Q10 increased further in response to 24 h cold acclimation is in accordance to Q10 values determined for adult zebrafish which are typically in the range between 4-6 . Therefore it can be expected that the highly energy consuming vertebrate brain ,, is exposed to acute energy fluctuations as well. Thus it is interesting and important to understand how brain energy metabolism of ectotherms is regulated under hypothermic stress. Previous studies demonstrated that Antarctic fish have higher lactate dehydrogenase (LDH) and citrate synthase (CS) activity in brain tissues compared to tropical and subtropical fishes ,. This suggests that despite comparatively lower metabolic rates and depressed enzymatic activities in locomotory muscle of cold adapted fish, the energy providing pathways that fuel metabolic demands of the brain have to compensate for cold-dependent activity losses ,. This ability is essential for brain functionality under hypothermic stress and allows fish to survive at low temperatures. On one hand, earlier studies indicated that fish can use lactate as an additional energy substrate during acclimation to severe environmental conditions including salinity and temperature changes ,,. On the other hand, lactate utilization is involved in glucose-sensing capacity and further allowed the maintenance of energy homeostasis in fish brain ,. In the present study, a significant 2-fold decrease, of brain lactate content was found in zebrafish brain transferred from 28°C to 18°C for 1 and 24 h. This decrease of lactate concentrations may indicate enhanced lactate utilization from the lactate pool in fish brain under hypothermic conditions. Together with increased LDH protein concentrations we suggest that lactate is metabolized at higher rates in response to acute cold stress in brain tissues. Kawall and colleagues  also denoted that LDH activities in Antarctic fish brains were significant higher compared to those in tropical/subtropical fish species. Therefore lactate utilization and LDH activation for ATP production may represent a potential benefit for energy mobilization in fish brain under hypothermic conditions. Moreover, increased protein levels of pace-determining metabolic enzymes involved in lactate metabolism and energy equivalent synthesis within the krebs cycle also support our hypothesis that lactate utilization is increased to control energy homeostasis in zebrafish brain during acute (1 and 24 h) cold stress. Our results further suggest that the “lactate-shuttle hypothesis” model established in mammalian systems has also evolved in the teleost brain . This hypothesis denotes that lactate formed in astrocytes is shuttled into neurons via MCTs as an alternative energy source. Thus, increased mRNA levels of PDP and PK particularly support this pathway because lactate transported into neurons needs to be converted to pyruvate and NADH. In the context of acclimation to cold stress, lactate may represent an important metabolite to support energy metabolism in fish brain. Ivanov and colleagues  demonstrated that lactate is an efficient energy substrate to fuel brain aerobic energy metabolism in case of insufficient glucose supply under intense synaptic activity. In this example LDH1 converts lactate to pyruvate and generates cellular NADH pools which represent an important physiological response to neural activation. This lactate metabolizing pathway has been suggested to be more beneficial for rapid energy supply compared to pyruvate formation via glycolysis . Although lactate has long been considered as a potentially toxic metabolic waste product recent findings could demonstrate that neurons preferentially consume lactate as energy substrate using rat neuronal culture experiments ,. In addition, lactate also evokes neuroprotective effects via transcriptional activation of brain-derived neurotrophic factor (BDNF), an essential factor for nerve cells survival . Based on our acute hypothermic challenge in zebrafish, we provided convincing molecular evidences to prove that the astrocyte-neuron lactate shuttle hypothesis (ANLSH) that has been proposed in mammals and other teleosts is an important pathway in fish brain, as well. The existence of the lactate shuttle in fish brain mediated by MCTs further suggests that lactate may serve as an alternative metabolite in brain tissues . Furthermore, transport of lactate between astrocytes and neurons via MCTs in ectothermic fish brain not only plays a critical role for glucosensing function , but is also beneficial for fish to cope with acute hypothermic stress.
In contrast to mammals, where only four MCT isoforms have been described, more additional paralogues have been found in teleosts. Some of these novel isoforms were demonstrated to mediate lactate cycling between cells in fish swim bladder . In this study, three novel MCT isoforms, drMCT1b, -2b and -4b, were explored and sequenced from zebrafish and most of them specifically clustered with those from other teleosts. Phylogenetic analysis inferred that diverse functionalities of MCT homologues could have evolved among teleosts and other higher vertebrates. Moreover according to the analysis of physicochemical properties, amino acid residues and membrane-spanning domains, all the drMCT paralogues were predicted to have a long C-terminus, and drMCT2b, -3, -4a and -4b have conventional features of 12 putative TMDs similar to MCT homologues of mammals ,. Interestingly only 11 putative TMDs were predicted for drMCT1a, -1b and -2a which differs from other homologues . According to findings in mammalian MCT structural characterizations, the greatest sequence variation between different MCT isoforms has been observed in the large cytoplasmic loop between TMDs 6 and 7 ; therefore it would be interesting to investigate whether structural differences in TMD numbers or interval loop between TMDs can be related to differential lactate transport abilities of MCTs from ectothermic animals. To offer more definite conclusions regarding structural and functional characteristics of MCT isoforms deeper protein structural analysis such as protein chimera studies, nuclear magnetic resonance spectroscopic (NMRS) or X-ray spectroscopic investigations in combination with determinations of lactate transport rates are needed.
Based on our semi-quantitative PCR results, each organ tested expressed more than one MCT isoforms in zebrafish with all seven MCT isoforms detected in brain tissues. In mammals, MCT1 was expressed in most of tissues and sometimes in combination with other MCT isoforms –. However, compared to MCT1, a higher affinity for lactate and pyruvate was found for MCT2 in mammalian systems . This paralogue is primarily expressed in liver, kidney, brain, sperm tail, skeletal muscle, and heart and has been described to transport significant amounts of lactate that can be used as energy source ,. In zebrafish, drMCT2a (annotated as zMCT2 in the previous study) has been previously described as an essential component for the development of the central nervous system (CNS) by mediating monocarboxylate transport . Compared with our previous study, the novel drmct2b orthologues were found to be specifically expressed in neurons while drmct2a was localized both in neuron and astrocyte  and expressed at higher rates than drmct2b. MCT3 has a unique distribution in the basal membrane of the mouse choroid plexus epithelia . Nevertheless in this study, drmct3 was highly expressed in most examined tissues except for liver, different from the mammalian MCT3 which was expressed in retinal cells only ,,. However, the physiological role of MCT3 remains unclear in the mammalian homologue . The fact that all the drMCT isoforms were expressed in different cell types of the zebrafish brain suggest that lactate transport between neuron and glial cells and its utilization are probably even more complex in ectothermic fish than in warm-blooded vertebrates.
In zebrafish brain, mRNA expressions of drmct1b and drmct4a were about 10-folds higher than that of drmct3, while drmct2a and drmct2b expression levels were comparatively lower than those of drmct3. This is different from findings in mammals where the expression level of MCT2 was higher than that of MCT1 in brain tissues . Compared to other MCT homologues, expression of drmct2a transcripts in fish brain was up-regulated both at 1- and 24-h cold shock, while the transcript of drmct4a was only increased after 24 h hypothermic stress. In contrast drmct1b/-2b (specifically expressed in neurons) and drmct3/-4b (expressed both in neurons and astrocytes) mRNA levels in brain were down-regulated or remained unchanged during the acute cold-shock period. According to these differential and partly opposing expression patterns of drMCT isoforms in brain we hypothesize that during acute cold stress an increased lactate demand is mediated by a shift between different MCT isoforms. In order to provide a deeper insight regarding the differential functionality of MCT isoforms we conducted lactate uptake experiments using MCT expressing oocytes.
Comparison of the expression and function of drMCT2aand-4bunder acute cold exposure in zebrafish
Fold of change
Fold of change
(for 24 h)
(for 24 h)
Relative expression (normalized by drrpl13a)
Lactate uptake rate (n mole/h/oocyte)
(A x B)
Theoretical lactate uptake capacity
Materials and methods
The wild type AB strain of adult zebrafish (Danio rerio) were obtained from the stock of the Institute of Cellular and Organismic Biology, Academia Sinica. Animals were kept in a circulating system at 28°C under 14 hour/10 hour of light/dark photoperiod. Fish were fed with dry food (Hai Feng, Nantou, Taiwan) twice a day.
Imported mature female African X. laevis frogs were purchased from the African Xenopus facility c.c., Noordhoek, South Africa. They were housed in cages in 18°C carbon-filtered water under standard conditions, and were fed small-sized live goldfish. Through an abdominal small (<1 cm) incision, oocytes were isolated by a partial ovariectomy from female Xenopus anesthetized with 0.1% tricaine (3-aminobenzoic acid ethyl ester) on ice. The incision was sutured, and the animal was monitored during the recovery period before it was returned to its tank. Oocytes were maintained at 18°C in Barth’s solution containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM CaN2O6, 0.41 mM CaCl2, 0.82 mM MgSO4, and 15 mM HEPES (pH 7.6), with gentamycin (20 μg/mL). Oocytes were utilized within 2 days after sampling.
Zebrafish acclimated in 28°C for over than six months and were directly transferred to 18°C circulating tanks. 8 males and 8 females (body length: 4.0~4.5 cm) were collected and both incubated in 5 L aquaria which were placed in a 300 L water bath at a constant temperature of 18 ± 0.5°C. The four replicate tanks were randomly distributed within the water bath and connected to a flow through system providing freshwater. After 1 h and 24 h cold transferred, fish were anesthetized with buffered MS222 (Sigma, St. Louis, MO, USA) before sampling and brains were sampled for total RNA, protein extraction and glucose/lactate content analysis. They were always sacrificed at the same time between 11:00 AM to 12:00 AM, in order to minimize the effect of circadian rhythm. In addition, our preliminary test could demonstrate that oxygen consumption rates were not affected by food deprivation for 24 h at 28°C. Therefore during the acclimation experiments, fish were not fed.
Oxygen consumption and NH4+ excretion rates
Oxygen consumption rates were determined by closed respirometry in 2 L respiration chambers at atmospheric pressure. Three chambers were filled with filtered freshwater (0.2 μm) and equipped with three animals each. An additional chamber without fish served as a control. Fish were pre acclimated in the respiration chamber for 2-h while the water was aerated to reach full oxygen saturation. After closing the respiration chambers animals were incubated for 1 h. In order to keep the incubation temperature constant, test chambers were placed in a temperature controlled water bath. A decrease of oxygen concentrations below 70% was avoided. Before and after incubation, water samples (3 × 50 ml plus overflow) were carefully siphoned off from each respiration chamber, and the amount of dissolved oxygen was determined following the Winkler method . The amount of oxygen consumed by bacteria (control chamber) was subtracted from the overall oxygen consumption of the fish. Oxygen consumption rates were expressed as μmole/mg FW *h.
To assess the effect of hypothermic treatment on ammonia excretion, one fish was placed in a 3 L tank, and 400 μL of water samples were be collected from the incubation water at the start of the experiment and after 1- and 24-h incubation respectively. Water samples were deproteinized in 4 volumes of ice-cold 6% perchloric acid, and centrifuged at 16000 g for 15 min at 4°C. The supernatant was further neutralized with ice-cold 2 M K2CO3 and spun again at 16000 g for 10 min (4°C). The final supernatant was directly analyzed for NH4+ concentrations using a tissue ammonia kit (AA0100; Sigma, St Louis, MO, USA). Ammonia contents in the incubation water were determined colorimetrically at an absorbance of 390 nm with a Synergy HT spectrophotometer (BIO-TEK, Winooski, Vermont, VT, USA).
Glucose and L-lactate content analysis
Isolated brain tissues were homogenized by polytron disruption with 7.5 volume of ice-cooled 6% perchloric acid (PCA), and neutralized (using 1 M potassium bicarbonate). The homogenate was centrifuged at 1000 × g for 15 min in 4°C, and the supernatant was used for the following assay. Glucose and lactate levels in zebrafish brain were assessed using the glucose and lactate assay kit (BioVision, Mountain View, CA, USA) following the manufacturer's protocols. Both, glucose and lactate contents were measured at 570 nm with a Synergy HT spectrophotometer (BIO-TEK) for colorimetric assay. Each sample was assayed in triplicate.
Isolated brain tissues were disrupted in homogenization buffer (100 mM imidazole, 5 mM EDTA, 200 mM sucrose, and 0.1% sodium deoxycholate; pH 7.6), and then centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant (a volume equivalent to 20 μg protein) was supplemented with electrophoresis sample buffer (250 mM Tris-base, 2 mM Na2EDTA, 2% SDS, and 5% dithiothreitol), and then incubated at 95°C for 10 min. The denatured samples were subjected to 10% sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, CA, USA). After blocking in 5% nonfat milk, the blots were incubated with a mouse anti-human lactate dehydrogenase 1 (LDH1) monoclonal antibody (Acris Antidodies GmbH, Germany, diluted 1:1000), a mouse anti-porcine citrate synthase (CS) monoclonal antibody (US Biological, Massachusetts, MA, USA, diluted 1:1000) and a mouse anti-chicken α-tubulin (Sigma), respectively. Blots were then incubated with alkaline phosphatase (AP)-conjugated goat anti-mouse immunoglobulin G (Pierce, Rockford, IL, USA, diluted 1:1000) for one hour. The immunoreactive proteins were visualized with a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBP) substrate kit for AP (Zymed Laboratories, San Francisco, CA, USA). Immunoblots were scanned and exported to TIFF files, and the differences between the band intensities of control fishes and cold acclimation fishes were compared using a commercial software package (Image-Pro Plus 7.0, Media Cybernetics, Silver Spring, MD, USA).
Preparation of total RNA
250-300 mg brain tissue were collected and homogenized in 4 mL of Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Genomic DNA was removed by treating total RNA with DNase I (Promega, Madison, WI, USA) at 37°C for 15 min, and then total RNA was purified by a RNA purification kit (MasterPureTM, EPICENTRE Biotechnologies, Madison, WI, USA). The amount and quality of total RNA were determined by measuring the absorbance at 260 nm and 280 nm with NanoDrop spectrophotometer (ND-1000, NadroDrop Technologies, Wilmington, Delaware, USA). Then the completeness of total RNA was checked by RNA denatured gels. The total RNA pellets were stored at -20°C.
Cloning, phylogenetic analysis and transmembrane prediction
In-silico predicted full-length of zebrafish (D. rerio) MCT (drmct) homologues obtained from the genome were carefully confirmed by the NCBI database. Specific primers (as listed in Additional file 1: Table S1) were designed for the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. PCR products were subcloned into a pGEM-T Easy vector (Promega, Madison, WI, USA), and the nucleotide sequences were determined with an ABI 377 sequencer (Applied Biosystems, Warrington, UK). Sequence analysis was conducted with a BLASTx program (NCBI).
Summary of the known and predicted homologs of solute carrier 16A (SLC16A) protein family
Scaffold GL831365.1: 0.77m
Scaffold GL831272.1: 0.54m
Scaffold GL173000.1: 0.36m
Scaffold Zv9_NA123: 35,158
Scaffold GL831137.1: 4.3m
Scaffold GL831152.1: 4.9m
Scaffold GL172918.1: 1.0m
Scaffold GL831179.1: 0.76m
Scaffold GL831279.1: 1.7m
Protein transmembrane predictions were generated using the “HMMTOP” program (http://www.enzim.hu/hmmtop/). On the basis of physicochemical properties (i.e. hydrophobicity, charges, and distribution) of drMCT paralogues’ amino acid residues and membrane-spanning features, the two-dimensional model images were created by a TMRPres2D software (http://biophysics.biol.uoa.gr/TMRPres2D/).
Reverse transcription-PCR analysis
Total RNA extracted from brain, eye, gill, heart, stomach, intestine, spleen, liver, kidney, testis, ovary and muscle of zebrafish were diluted to equal concentration as template for reverse transcription (5 μg total RNA/reaction). The final volume of 20 μL contained 0.5 mM dNTPs, 2.5 μM oligo(dT)20 primer, 5 mM dithiothreitol, and 200 units PowerScript reverse transcriptase III (Invitrogen, Carlsbad, CA, USA) and was incubated for 90 min at 50°C followed by 15 min incubation at 70°C. Then 20 units Escherichia coli RNase H (Invitrogen, Carlsbad, CA, USA) was added to remove the remnant RNA. For PCR amplification, 2 μL total cDNA was used as template in a 50 μL final reaction volume containing 0.25 mM dNTPs, 2 units ExTaq polymerase (Takara, Shiga, Japan), and 0.2 μM of each primer. Primers were designed against specific regions of each isoform according to the drMCT alignment results (Figure 6). The primer sets for the PCR are shown in Additional file 1: Table S1. Zebrafish ribosomal protein L13A (zrpl13a) was used to evaluate the relative amounts of cDNAs as an internal control. This gene has been demonstrated to serve as a suitable reference gene in zebrafish under diverse experimental treatments and different developmental periods . All amplicons were sequenced to ensure that PCR products corresponded to the desired gene fragments.
Quantitative real-time PCR (qRT-PCR) analysis of gene expressions
Total RNA was extracted and reverse-transcribed from zebrafish brain as described above. Real-time PCR was performed with Roche LightCycler® 480 System (Roche Applied Science, Mannheim, Germany). The primers for all genes were designed (Additional file 1: Table S1) using Primer Premier 5.0 software (PREMIER Biosoft Int., Palo Alto, CA, USA). The final volume of 10 μL containing 40 ng of cDNA, 50 nM of each primer and the LightCycler® 480 SYBR Green I Master (Roche). The standard curve of each gene was checked in linear range with drrpl13a as an internal control. All qRT-PCR reactions were administered as follows: 1 cycle of 50°C for 2 min and 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min. PCR products were subjected to melting-curve analysis, and representative samples were electrophoresed to verify that only a single product was present. All primer pairs used in this PCR had efficiencies >94%. Control reactions were conducted with sterile water to determine the levels of the background and genomic DNA contamination.
Fluorescent in situ hybridization and immunocytochemical staining
Digoxigenin (DIG)-labeled RNA probes of drmct1b, drmct2b and drmct4b sense and antisense strands were synthesized by in vitro transcription using DIG-labeling mix (Roche, Grenzach-Wyhlen, Germany) according to the manufacturer’s instructions. RNA probes were examined on RNA gels.
The brain was dissected out of the adult zebrafish head capsule and then sliced into transverse sections of 50 μm using a vibrating blade microtome (VT–1200S, Leica, Wetzlar, Germany). Sliced brain samples were attached to poly-L-lysine-coated slides (Erie, Portsmouth, NH, USA). Prepared slides were washed with PBST and then hybridized with prepared probes at 65°C for overnight incubation. On the next day, slides were washed in a hybridization buffer series in 2× SSC at 65°C. After another serial washing with 0.2× SSC and PBST, samples were incubated with 5% sheep serum in 2 mg/mL bovine serum albumin (BSA), and then the anti-DIG-POD antibody (Roche) was added at a 1:500 dilution. Slides were then washed with PBST and stained with the Alexa 488-tyramide substrate (1:100 dilution). After being washed with PBST and 100% methanol, slides were incubated in an H2O2/methanol solution to inactivate the POD. The validity of antisense RNA probes’ signals was further examined by the same experiments utilizing sense probes (data not shown). After in situ hybridization, zebrafish brain slides were rinsed in PBS, and blocked with 3% BSA. Afterwards, slides first incubated with the ZN12 monoclonal antibody (Institute of Neuroscience, University of Oregon, Eugene, OR, USA; diluted 1:100) to label neurons, or with the glial fibrillary acidic protein (GFAP) mAb (DAKO, Glostrup, Denmark; diluted 1:100) to label astrocytes. After washing with PBS, slides were incubated in anti-mouse IgG conjugated with Alexa Fluor 568 (1:300; Molecular Probes, Eugene, OR, USA). Then slides were washed with PBS. Images were acquired with a Leica TCS-SP5 confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany).
Synthesis of drmct homologue capped mRNA (cRNA)
The open-reading frame of drmct homologues and the designed anchor sites (XhoI and BamHI) were PCR-amplified using primers based on gene coding sequence, including its stop codon. They were cloned into a pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced. The PCR products were then digested with XhoI and BamHI and subcloned in-frame into the pCS2XLT vector, in which the signal sequence was not fused with the N-terminus of the green fluorescent protein GFP. Capped mRNA (cRNA) encoding drmct paralogues were transcribed using the SP6 mMessage mMachine (Ambion, Austin, TX, USA) from plasmid template linearized with NotI (Promega, Madison, WI, USA).
Expression of drmct homologues in X. laevis oocytes
5 ng of drmct homologue cRNA was microinjected into stage V-VI Xenopus oocytes. An equal volume of RNase-free modified Barth’s saline (MBS, containing 85 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 10 mM HEPES, 3 mM NaOH, pH 7.4, supplemented with 10 units/ml penicillin and 10 μg/ml streptomycin) was also injected to serve as the control group. After culturing for 2 days at 18°C MBS, the uptake experiment was initiated by transferring and incubating the oocytes at 18°C or 28°C in 500 μL MBS medium for 6 h and then 5 kBq/ml of L-[14C]-lactate (Amersham, Piscataway, NJ, USA) was added into the MBS medium. After incubation in L-[14C]-lactate containing MBS medium for 2 h, the uptake reaction was terminated by adding 2 mL of ice-cold MBS, followed by washing the oocytes five times with 2 mL MBS. After the wash, each oocyte was dissolved with 300 μL of 10% SDS. Radioactivity was determined by adding 2 mL of ACSII (Amersham) to each solubilized oocyte in a liquid scintillation counter and then total L-[14C]-lactate concentration in experimental oocytes was determined in a beta counter (LS6500 Liquid Scintillation Counter, Beckman Coulter, Fullerton, CA, USA). The lactate uptake rate of MBS injected oocytes is subtracted from the uptake rate of MCT expressing oocytes and presented as nmole/hr per oocyte.
Values are presented as mean ± standard deviation (SD). The level of significance was set to p < 0.05 in a one-way ANOVA (Tukey’s pairwise comparison) for calculating intact O2 consumption and ammonium excretions of zebrafish; glucose and L-lactate contents in brain; LDH1 protein relative abundance; homologues of drpk, drpdp and drmct mRNA expression levels; L-[14C]-lactate transport characterizations of drMCT2a and drMCT4b for each time point along cold treatment. Student's t-test was used for analyzing ammonium concentration, CS protein expression and L-[14C]-lactate uptake between control (28°C) and hypothermic (18°C) groups. Different letters indicate significant differences between treatments (p < 0.05), whereas same letters indicate no significant differences between treatments (p > 0.05). Asterisks indicate significant different between control and hypothermic group (Student’s t-test, p < 0.05).
This study was financially supported by the grants to Y. C. Tseng from the National Science Council, Taiwan, Republic of China (NSC 102-2321-B-003 -002) and an Alexander von Humbold/National Science Council (Taiwan) grant awarded to M. H. (NSC 102-2911-I-001-002-2).
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